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High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease.
Title | High-throughput protein production and purification at the Seattle Structural Genomics Center for Infectious Disease. |
Publication Type | Journal Article |
Year of Publication | 2011 |
Authors | Bryan, CM, Bhandari, J, Napuli, AJ, Leibly, DJ, Choi, R, Kelley, A, Van Voorhis, WC, Edwards, TE, Stewart, LJ |
Journal | Acta Crystallogr Sect F Struct Biol Cryst Commun |
Volume | 67 |
Issue | Pt 9 |
Pagination | 1010-4 |
Date Published | 2011 Sep 1 |
ISSN | 1744-3091 |
Keywords | Communicable Diseases, Genomics, Proteins |
Abstract | The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100 s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography. |
DOI | 10.1107/S1744309111018367 |
Alternate Journal | Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. |
PubMed ID | 21904042 |